ES2277581T3 - PROTEINS AND PEPTIDES ALLERGEN FROM DOMESTIC POWDER ACAROS AND USES OF THE SAME. - Google Patents

Abstract

Isolated nucleic acid comprising a nucleotide sequence encoding a Der f VII protein allergen comprising the amino acid sequence of SEQ ID NO: 7 or the mature portion of said allergen.

Description

Mite allergen proteins and peptides from household dust and uses thereof.

Background of the invention

Individuals of approximately 10% of the population become hypersensitized (allergic) upon exposure to antigens from a variety of environmental sources. Those antigens that induce immediate and / or delayed types of hypersensitivity are known as allergens (King, TP, (1976) Adv. Immunol ., 23 : 77-105). These include grass products, trees, weeds, animal dander, insects, food, drugs and chemicals. It is believed that the genetic predisposition of an individual plays a role in the development of immediate allergic responses (Young, RP et al ., (1990) Clin. Sci ., 79 : 19) such as atopy and anaphylaxis whose symptoms include fever Hay, asthma and hives.

The antibodies involved in atopic allergy belong mainly to the IgE class of immunoglobulins. IgE is fixed to basophils, mast cells and dendritic cells through a specific high affinity receptor Fc? (Kinet, JP, (1990) Curr. Opin. Immunol ., 2 : 499-505). When the combination of an allergen that acts as a ligand with its related receptor IgE takes place, the FceRI bound to the IgE can be subject to intermolecular binding on the cell surface, resulting in physiological manifestations of the interaction between the allergen and the IgE These physiological effects include the release of, among other substances, histamine, serotonin, heparin and fac-
chemotactic tor (s) for eosinophilic leukocytes and / or C4, D4 and E4 leukotrienes, which cause prolonged constriction of bronchial smooth muscle cells (Hood, LE et al ., Immunology (2nd ed.), The Benjamin / Cumming Publishing Co., Inc. (1984)). Therefore, the last consequence of the interaction of an allergen with IgE is that it consists in the appearance of allergic symptoms activated by the release of the mediators mentioned above. Such symptoms may be systemic or local in nature, depending on the route of entry of the antigen and the pattern of IgE deposition on mast cells or basophils. Local manifestations generally take place on epithelial surfaces at the site of allergen entry. Systemic effects can induce anaphylaxis (anaphylactic shock) that results from the response of the IgE-basophil combination to the circulating (intravascular) antigen.

Studies with purified allergens have shown that approximately 80% of patients who are allergic to the Dermatophagoides pteronyssinus mite produce IgE reactive to Der p I and Der p II (Chapman MD et al ., J. Immunol . (1980 ) 125 : 587-92; Lind P., J. Allergy Clin. Immunol . (1985) 76 : 753-61; Van derZee JS et al ., J. Allergy Clin. Immunol . (1988) 81 : 884-95) . For approximately half of the patients, these specificities constitute 50% of the IgE anti-mite antibody. The allergen Der p III, recently identified as trypsin, (Stewart GA et al ., Immunology (1992) 75 : 29-35) reacts with a similar or higher frequency (Stewart GA et al ., Supra ; Ford SA et al . , Clin. Exp. Allergy (1989) 20 : 27-31). However, in the only quantitative study conducted to date, the researchers reported that the level of IgE fixation is considerably lower than that of Der p I. Electrophoresis techniques (Ford SA et al ., Supra ; Bengtsson A . et al ., Int. Arch. Allergy Appl. Immunol . (1986) 80 : 383-90; Lind P. et al ., Scand. J. Immunol . (1983) 17 : 263-73; Tovey ER et al . J. Allergy Clin. Immunol . (1987) 79 : 93-102) have shown that most sera recognize other allergens. For example, in the study by Ford et al . ( supra ) Western blot analysis showed 8 sera reacting with 1-2 bands, 6 with 3-6 and 3 with a larger number, including one with at least 13. In another study, Baldo et al . ( Adv. Bioscience (1989) 4 : 13: 31) report on the finding of components at the level of a Mr of 30, 26 and 25 K that react with 50% of sera. To determine the importance of particular specificities in allergic reactions, purified allergens would be required for quantitative IgE fixation assays and to examine the frequency and lymphokine profile for the reactivity of T cells.

The treatment of sensitive patients to house dust mites by dose administration Increasing domestic dust extracts has the drawbacks of the potential anaphylaxis during treatment and of the possible need to continue therapy for a period of time of several years to generate a sufficient tolerance that results in a significant decrease in clinical symptoms. A therapeutic composition and method would be beneficial therapeutic to avoid these problems.

Brief exposition of the invention

This invention provides isolated nucleic acids encoding peptides that have the antigenic activity of Der f VII, which is a protein allergen of the Dermatophagoides pharynx species. The preferred nucleic acid is cDNA (cDNA = complementary DNA) having a nucleotide sequence illustrated in Figure 6A and 6B (SEQ ID NO: 6) ( Der f VII). The invention is also related to peptides that are encoded throughout this cDNA (SEQ ID NO: 6) or a part thereof and that have the antigenic activity of Der f VII. Nucleic acids that hybridize under strict conditions are also contemplated (ie, equivalent to about 20-27 ° C below the melting temperature (T m) and about 1M salt) to form a nucleic acid with a nucleotide sequence illustrated in Figure 6A and 6B (SEQ ID NO: 6) or encoding a peptide comprising all or a portion of an amino acid sequence of Figure 6A and 6B (SEQ ID NO: 7) ( Der f VII). Nucleic acids encoding peptides that have a Der f VII activity and have at least 50% homology are also provided with a sequence illustrated in Figure 6A and 6B (SEQ ID NO: 7) ( Der f VII). Peptides having a Der f VII activity produced by means of the recombinant expression of a nucleic acid of the invention are also provided by this invention, and peptides having a Der f VII activity prepared by chemical synthesis. Preferred peptides have the ability to induce a T cell response that may include stimulation of T cells (measured, for example, by proliferation of T cells or cytokine secretion) or inability to respond to T cells (that is, contact with the peptide or with a peptide complex with an MHC molecule (MHC = major histocompatibility complex) of a cell presenting the antigen induces the T cell to be unable to respond to stimulatory signals or being unable to proliferate). Other preferred peptides, either apart from or in addition to the ability to induce a T-cell response, have the ability to fix the specific IgE of house dust mites of subjects allergic to dust mites. Such peptides are useful for diagnosing the sensitivity of a subject to dust mites. Even other peptides, either apart or in addition to the ability to induce a T-cell response, have a considerably reduced ability to fix allergic IgE to dust mites. Such peptides are particularly useful as therapeutic agents.

Other preferred peptides comprise an amino acid sequence illustrated in Figure 6A and 6B (SEQ ID NO: 7) ( Der f VII). In one embodiment, peptides are provided that have Der f VII antigenic activity and comprise a part of the amino acid sequence of Figure 6A and 6B (SEQ ID NO: 7). Such peptides are at least 8-30 amino acids in length, preferably 10-20 amino acids in length, and most preferably, 10-16 amino acids in length.

Another aspect of the invention provides antibodies that are specifically reactive with a peptide having antigenic activity of Der f VII. A peptide having the antigenic activity of Der f VII can be used in compositions suitable for pharmaceutical administration. Such compositions can be used in a manner similar to how dust mite extracts are used to treat or prevent allergic reactions to an allergen from dust mites in a subject. They can also be used to diagnose the sensitivity of a subject to an allergen in the dust mites nucleic acids of the invention and peptides having the antigenic activity of Der f VII.

Brief description of the drawings

Fig. 1 illustrates the fixing frequency of IgE of allergic sera with plaques λ11-HD6.

Fig. 2 shows the reactivity of IgE and rabbit dust mite antibody against domestic HD6 insert fusion product cloned into pGEX-1 glutathione-S-transferase purified.

Fig. 3A and Fig. 3B is the nucleotide sequence and the deduced amino acid sequence of the Der p VII HD6 clone.

Fig. 4 shows extracts of house dust mites characterized by electrophoresis in an SDS-PAGE (SDS-PAGE = characterization procedure by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) at 8-18%, with electrotransfer to nitrocellulose and Combined allergic serum reaction absorbed with E. coli lysates containing a vector control pGEX-1 ( lane 1 ) or HD6 of pGEX-1 ( lane 2 ).

Fig. 5 shows the reactivity of anti-HD6 antibodies purified according to affinity for extracts of D. Pteronyssinus . Rabbit antibodies were purified according to affinity on nitrocellulose, and were used to serve as a probe in a Western blot analysis of mite extracts, with electrophoresis in 8-18% SDS-PAGE, and were revealed with Protein A -125 I.

Fig. 6A and Fig. 6B is the nucleotide sequence and the deduced amino acid sequence of Der f VII.

Fig. 7A, 7B, 7C, 7D and 7E is a comparison of the nucleotide sequence and the deduced amino acid sequence between Der f VII and Der p VII. The dots indicate equality in the nucleotide sequence Der f VII and Der p VII. Nucleotide bases that differ between Der f VII and Der p VII are indicated, along with any difference in the corresponding amino acids.

Detailed description of the invention

This invention is related to isolated nucleic acids encoding peptides having the antigenic activity of Der f VII, which is an allergen of the Dermatophagoides farinae species. Preferably, the nucleic acid is a cDNA comprising a nucleotide sequence illustrated in Figure 6A and 6B (SEQ ID NO: 6) ( Der f VII).

The cDNA illustrated in Figure 6A and 6B (SEQ ID NO: 6) encodes a Der f VII peptide. Said Der f VII peptide is encoded by bases 43 to 681 of this cDNA sequence. The deduced amino acid sequence of Der f VII based on this cDNA is also illustrated in Figure 6A and 6B (SEQ ID NO: 7). Said Der f VII peptide may contain a head sequence not found in the mature protein.

A host cell transfected with an expression vector containing a nucleotide sequence encoding Der f VII was deposited according to the Budapest Treaty at the Australian State Analytical Laboratories on May 10, 1994, and said cell was given the number of entry register N94 / 8705.

Accordingly, one aspect of this invention is related to isolated nucleic acids comprising nucleotide sequences encoding Der f VII, fragments thereof encoding peptides having the antigenic activity of Der f VII, and equivalent of such nucleic acids. In the sense that it is used herein, it is understood that the term "nucleic acid" includes such fragments and equivalents. The term "equivalent" is used with the intention of including nucleotide sequences encoding functionally equivalent Der f VII proteins or functionally equivalent peptides having the antigenic activity of Der f VII. In the sense defined herein, a peptide having a Der f VII activity has at least one biological activity of the Der f VII allergen. The equivalent nucleotide sequences will include sequences that differ in one or more substitutions, additions or nucleotide deletions, such as allelic variants, and will also include sequences that differ from the nucleotide sequence encoding Der f VII illustrated in Figure 6A and 6B (SEQ ID NO. : 6) due to the degeneracy of the genetic code. The equivalents will also include nucleotide sequences that hybridize under strict conditions (i.e., equivalent to approximately 20-27 ° C below the melting temperature (T m) and approximately 1M salt) to form the nucleotide sequence of Der f VII illustrated in Figure 6A and 6B (SEQ ID NO: 6).

Peptides referred to herein as peptides that have Der f VII antigenic activity or that have Der f VII activity are defined herein as peptides having an amino acid sequence that substantially corresponds to the totality. or to a part of the amino acid sequence of Der f VII illustrated in Figure 6A and 6B (SEQ ID NO: 7), whose peptide has the antigenic activity of Der f VII. For example, a peptide having the antigenic activity of Der f VII may have the ability to induce a response in T cells restricted by Der f VII, such as stimulation (eg T cell proliferation or cytokine secretion ), or to induce the inability to respond to T cells. Alternatively, or additionally, a peptide having a Der f VII activity may have the ability to bind (if recognized by) immunoglobulin E (IgE) antibodies of subjects allergic to dust mites. Peptides that bind IgE are useful in methods to detect allergic sensitivity to Der f VII in a subject. Peptides that do not bind IgE, or that bind IgE to a lesser extent compared to the degree to which a purified native Der f VII protein binds IgE, are particularly useful as therapeutic agents.

In one embodiment, the nucleic acid is a cDNA encoding a peptide that has the antigenic activity of Der f VII. Preferably, the nucleic acid is a cDNA molecule comprising at least a part of the nucleotide sequence encoding Der f VII illustrated in Figure 6A and 6B (SEQ ID NO: 6). A preferred part of the cDNA molecules of Figure 6A and 6B includes the coding region of the molecule.

In another embodiment, the nucleic acid of the invention encodes a peptide that has the antigenic activity of Der f VII, and comprises an amino acid sequence illustrated in Figure 6A and 6B (SEQ ID NO: 7) ( Der f VII). Preferred nucleic acids encode a peptide having Der f VII antigenic activity and at least about 50%, more preferably at least about 60%, and most preferably at least about 70%, homology with a sequence set forth in Figure 6A and 6B (SEQ ID NO: 6) ( Der f VII). Nucleic acids encoding peptides that have Der f VII antigenic activity and have at least about 90%, more preferably at least about 95%, and most preferably at least about one within the scope of the invention are also within the scope of the invention. 98-99%, homology with a sequence set out in Figure 6A and 6B (SEQ ID NO: 7) ( Der f VII). The term homology refers to the similarity of sequences between two peptides that have a Der f VII activity or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, whose sequences can be aligned for comparative purposes. When a position in the compared sequence is occupied by the same base or by the same amino acid, the molecules are then homologous in that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

Another aspect of the invention provides a nucleic acid that hybridizes under very strict conditions to form a nucleic acid encoding a peptide that has all or part of an amino acid sequence illustrated in Figure 6A and 6B (SEQ ID NO: 7) ( Der f VII). Appropriate requirement conditions that favor hybridization, such as 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by washing at 2.0 x SSC at 50 ° C, are well known to those skilled in the matter can be found in Current Protocols in Molecular Biology , John Wiley & Sons, NY (1989), 6.3.1-6.3.6. For example, the salt concentration in the washing step can be selected from a few strict conditions, from about 2.0 x SSC at 50 ° C, to very strict conditions, from about 0.2 x SSC at 50 ° C. In addition, the temperature in the washing stage may increase from a few strict conditions, at room temperature (approximately 22 ° C), to very strict conditions, at approximately 65 ° C.

Also within the scope of the invention are isolated nucleic acids encoding peptides that have Der f VII antigenic activity, as described herein, and which have a sequence that differs from the nucleotide sequences illustrated in Figure 6A and 6B. (SEQ ID NO: 6) due to degeneracy in the genetic code. Such nucleic acids encode functionally equivalent peptides (i.e., a peptide that has a Der f VII activity) but differ in terms of the sequence of the sequences of Figure 6A and 6B due to the degeneracy of the genetic code. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine), can result in "silent" mutations that do not affect the amino acid sequence of the Der f VII protein. However, it is expected that polymorphisms of the DNA sequence that result in modifications of the amino acid sequence of Der f VII exist within the dust mite population. One skilled in the art will be aware that these variations in one or more nucleotides (in up to about 3-4% of the nucleotides) of the nucleic acids encoding peptides having a Der f VII activity may exist between different mites. of dust due to natural allelic variation. All and any such variations of the nucleotides and the resulting polymorphisms of the amino acids are within the scope of this invention. In addition, there may be one or more isoforms or one or more related and interreactive members of the Der f VII family. Such isoforms or family members are defined as related proteins in terms of function and amino acid sequence with Der f VII but encoded by genes in different loci.

Fragments of the nucleic acid encoding Der f VII are also within the scope of the invention. In the sense that it is used herein, the term "nucleic acid fragment encoding Der f VII" refers to a nucleotide sequence that has fewer nucleotides than the nucleotide sequence encoding the entire sequence of amino acids of the Der f VII protein and encoding a peptide that has an activity of Der f VII (i.e., a peptide that has at least one biological activity of the Der f VII allergen) as defined herein.

Preferred nucleic acid fragments encode peptides of a length of at least about 7 amino acid residues, preferably of a length of about 13-40 amino acid residues, and more preferably, of a length of approximately 16-30 amino acid residues. . Also within the scope of this invention are nucleic acid fragments encoding peptides having a Der f VII activity of a length of at least about 30 amino acid residues, a length of at least about 40 amino acid residues, a length of at at least about 60 amino acid residues, a length of at least about 80 amino acid residues, a length of at least about 100 amino acid residues, a length of at least about 140 residues, and a length of at least about 200 residues or more. In general, the expression of the peptides in a transformed host cell is maximally advantageous when the desired peptide is more than about 20 amino acids in length. Shorter peptides are typically synthesized more readily by chemical means.

Nucleic acid fragments that are within the scope of the invention include those that are capable of hybridizing under very strict conditions with nucleic acids of other animal species to be used in screening protocols to detect Der f VII or allergens that interact with Der f VII. Generally, the nucleic acid encoding a peptide having a Der f VII activity will be selected from the bases encoding the mature protein, although in some cases it may be desirable to select all or part of a peptide from the part of the head sequence of the nucleic acids of the invention. Nucleic acids that are within the scope of the invention may also contain binding sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of recombinant peptides having a Der f VII activity.

A nucleic acid encoding a peptide having a Der f VII activity can be obtained from mRNA (mRNA = messenger RNA) from the Dermatophagoides farinae dust mite. It should also be possible to obtain nucleic acids from the Dermatophagoides farinae genomic DNA encoding Der f VII. For example, the gene encoding Der f VII can be cloned either from a cDNA or from a genomic library according to the protocols described herein. A cDNA encoding Der f VII can be obtained based on isolating total mRNA from Dermatophagoides farinae . Then double stranded cDNAs can be prepared from the total mRNA. Subsequently, cDNAs can be inserted into a suitable plasmid or bacteriophage vector using any of a series of known techniques. Genes encoding Der f VII can also be cloned using accredited polymerase chain reaction techniques (see Examples 1 and 2) according to the nucleotide sequence information provided by the invention. The nucleic acids of the invention can be DNA or RNA. A preferred nucleic acid is a cDNA encoding Der f VII having the sequence illustrated in Figure 6A and 6B (SEQ ID NO: 6) ( Der f VII).

This invention also provides expression vectors containing a nucleic acid encoding a peptide having a Der f VII activity, operably linked to at least one regulatory sequence. The term "operably linked" is used to express that the nucleotide sequence is linked to a regulatory sequence in a manner that allows the expression of the nucleotide sequence. Regulatory sequences are recognized in the art and are selected to direct the expression of the peptide that has a Der f VII activity. Consequently, the term "regulatory sequence" includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185 , Academic Press, San Diego, CA (1990). It should be understood that the design of the expression vector may depend on factors such as the selection of the host cell to be transformed and / or the type of protein to be expressed. In one embodiment, the expression vector includes a DNA encoding a peptide having the antigenic activity of Der f VII. Such expression vectors can be used to transfect cells to thereby produce proteins or peptides, including fusion proteins or peptides encoded by nucleic acids described herein.

This invention is also relative to a transfected host cell to express a peptide having the antigenic activity of Der f VII. The host cell can be any prokaryotic or eukaryotic cell. For example, a peptide having the antigenic activity of Der f VII can be expressed in bacterial cells such as E. coli , insect cells (baculovirus), yeast or mammalian cells such as hamster ovary cells from China (CHO ). Other suitable host cells can be found in Goeddel, (1990) supra , or such cells are known to those skilled in the art.

Expression in eukaryotic cells, such as mammalian, yeast or insect cells, may result in partial or complete glycosylation and / or the formation of relevant interchain chains or intra-chain disulfide bonds of recombinant protein. Examples of vectors for S. cerivisae yeast expression include PYepSec1 (Baldari. Et al ., (1987) Embo J. , 6 : 229-234), pMFa (Kurjan and Herskowitz, (1982) Cell , 30: 933-943), pJRY88 (Schultz et al ., (1987) Gene , 54 : 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA). Baculovirus vectors available for protein expression in cultured insect cells (SF 9 cells) include the pAc series (Smith et al ., (1983) Mol. Cell Biol ., 3 : 2156-2165) and the pVL series ( Lucklow, VA and Summers, MD, (1989) Virology , 170 : 31-39). COS cells (Gluzman, Y., (1981) Cell , 23 : 175-182) are generally used in conjunction with vectors such as pCDM 8 (Aruffo, A. and Seed, B., (1987) Proc. Natl. Acad Sci. USA , 84 : 8573-8577) for transient amplification / expression in mammalian cells, while CHO (dhfr-Ovary Hamster of China) cells with vectors such as pMT2PC (Kaufman et al ., (1987) EMBO J. , 6 : 187-195) for stable amplification / expression in mammalian cells. The vector DNA can be introduced into mammalian cells by conventional techniques such as coprecipitation in calcium phosphate or calcium chloride, transfection mediated by DEAE-dextran (= diethylaminoethyldextran) or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al ., ( Molecular Cloning: A Laboratory Manual , 2nd Edition, Cold Spring Harbor Laboratory press (1989)) and in other laboratory textbooks.

Prokaryotic expression is most often carried out in E. coli with inducible fusion or non-fusion expression vectors. Fusion vectors usually add a number of amino acids with NH2 terminal to the expressed target gene. These amino acids with the NH2 terminal are often called the reporter group. Such reporter groups usually have the following two purposes: 1) increase the solubility of the target recombinant protein; and 2) assist in the purification of the target recombinant protein by acting as a ligand in the affinity purification. Often, a proteolytic segmentation site is introduced into the fusion expression vectors at the junction of the reporter group and the target recombinant protein to allow the target recombinant protein to separate from the reporter group after purification of the fusion protein . Such enzymes and their related recognition sequences include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Amrad Corp. Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase, binding protein of maltose E, or protein A, respectively, with the target recombinant protein.

Inducible non-fusion expression vectors include pTrc (Amann et al ., (1988) Gene , 69 : 301-315) and pET 11d (Studier et al ., Gene Expression Technology: Methods in Enzymology , 185 , Academic Press , San Diego, California (1990) 60-89). While the expression of the target genes is based on the transcription of host RNA polymerase from the trp-lac hybrid fusion promoter in the pTrc, the expression of the target genes inserted into the pET 11d is based on the transcription from the promoter of T7 gn10-lac 0 fusion mediated by coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by the BL21 (DE3) or HMS174 (DE3) host strains of a resident λ proago that hosts a T7 gn1 under the transcriptional control of the lacUV 5 promoter.

Our strategy to maximize the expression of recombinant Der f VII in E. coli is to express the protein in a host bacterium with an impaired ability to proteolytically segment the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology , 185 , Academic Press, San Diego, California (1990) 119-128). Another strategy is to alter the nucleic acid encoding the Der f VII protein to be inserted into an expression vector so that the individual codons for each amino acid are preferably used in highly expressed E. coli proteins (Wada et al . , (1992) Nuc. Acids Res ., 20 : 2111-2188). Such alteration of nucleic acids of the invention can be carried out by standard DNA synthesis techniques.

The nucleic acids of the invention can also be chemically synthesized using standard techniques. Several methods are known for chemically synthesizing polydeoxynucleotides, including solid phase synthesis, which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (see, e.g., Itakura et al . , US Patent No. 4,598,049; Caruthers et al ., US Patent 4,458,066; and Itakura, US Patent Nos. 4,401,796 and 4,373,071, incorporated herein by reference).

This invention is also related to methods for producing peptides having the antigenic activity of Der f VII. For example, a host cell transfected with a nucleic acid vector that directs the expression of a nucleotide sequence encoding a peptide that has the antigenic activity of Der f VII can be cultured under appropriate conditions to allow peptide expression to take place. . The peptide can be secreted and isolated from a mixture of cells and medium containing the peptide having the antigenic activity of Der f VII. Alternatively, the peptide can be retained cytoplasmically, and the cells can be collected and lysed, and the protein can be isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The peptide having the antigenic activity of Der f VII can be isolated from cell culture medium, host cells or both using techniques known in the art of protein purification, including ion exchange chromatography, filtration chromatography on gel, ultrafiltration, electrophoresis and purification according to immunoaffinity with antibodies specific to a peptide having an activity of Der f VII.

Another aspect of the invention is related to isolated peptides having the antigenic activity of Der f VII. A peptide having an activity of Der f VII has at least one biological activity of the Der f VII allergen. For example, a peptide having the antigenic activity of Der f VII may have the ability to induce a response in Der f VII specific T cells, such as stimulation (proliferation of T cells or cytokine secretion) or to induce inability to respond to T cells. In one embodiment, a peptide having a Der f VII activity stimulates T cells as evidenced, for example, by T cell proliferation or cytokine secretion. In another embodiment, peptides having the antigenic activity of Der f VII induce the inability of T cells to respond because T cells do not respond to a subsequent confrontation with a Der f VII peptide when exposed to the peptide. In yet another embodiment, a peptide having the antigenic activity of Der f VII has a reduced IgE binding activity compared to the purified native Der f VII protein. A peptide having a Der f VII activity may differ in terms of the amino acid sequence of the Der f VII sequence illustrated in Figure 6A and 6B (SEQ ID NO: 7) ( Der f VII), but such differences result in in a modified protein that functions in the same way or in a manner similar to that of the native Der f VII protein, or that has the same or similar characteristics as the native Der f VII protein. Various modifications of the Der f VII protein to produce these peptides and other functionally equivalent peptides are described in detail herein. In the sense in which it is used herein, the term "peptide" refers to full-length proteins and polypeptides or peptide fragments thereof.

A peptide may be produced by modification of the amino acid sequence of the Der f VII protein illustrated in Figure 6A and 6B (SEQ ID NO: 7) ( Der f VII), such as a substitution, addition or deletion of a residue of amino acids that is not directly involved in protein function. The peptides of the invention may be at least 10 amino acid residues in length, preferably approximately 10-20 amino acid residues, and more preferably, approximately 10-16 amino acid residues in length. Also included within the scope of this invention are peptides that have the antigenic activity of Der f VII and that are of a length of at least about 30 amino acid residues, of a length of at least about 40 amino acid residues, of a length of at least about 60 amino acid residues, of a length of at least about 80 amino acid residues, and of a length of at least about 100 amino acid residues.

Another embodiment of the invention provides a pure substance preparation of a peptide having the antigenic activity of Der f VII. Such a preparation is in substance free of the proteins and peptides with which the peptide occurs naturally (i.e., other peptides from dust mites) either in a cell or when secreted by a cell.

In the sense that it is used herein, the word "isolated" refers to a nucleic acid or peptide that is in substance free of cellular material or culture medium when produced by recombinant DNA techniques, or precursors. chemical or other chemical substances when chemically synthesized. Such proteins or peptides are also characterized by the fact that they are free from all other proteins in dust mites. Consequently, an isolated peptide having the antigenic activity of Der f VII is produced by recombination or synthesis and is in substance free of cellular material and culture medium or is in substance free of chemical precursors or other chemical substances and is in substance free of all other proteins from dust mites. An isolated nucleic acid is also free of the sequences that naturally flank the nucleic acid (that is, of the sequences located at the 5 'and 3' ends of the nucleic acid) in the organism from which the nucleic acid is derived.

Peptides having the antigenic activity of Der f VII can be obtained, for example, by selecting the peptides produced by recombination from the corresponding Der f VII nucleic acid fragment encoding such peptides. In addition, the fragments can be chemically synthesized using techniques known in the art such as conventional solid phase f-Moc or t-Boc chemistry of Merrifield. For example, the Der f VII protein may be arbitrarily divided into fragments of the desired length without overlapping the fragments, or it may preferably be divided into overlapping fragments of a desired length. The fragments can be produced (by recombination or by chemical synthesis) and can be analyzed to identify those peptides that have the activity of Der f VII (i.e., the ability to induce a T-cell response such as stimulation (proliferation, cytokine secretion), inability to respond and / or reduced IgE binding activity).

In one embodiment, peptides having the antigenic ability of Der f VII can be identified by the ability of the peptide to stimulate T cells or to induce the inability of T cells to respond. Peptides that stimulate T cells, as determined , for example, proliferation of T cells or cytokine secretion, are defined herein as peptides comprising at least one epitope for T cells. Epitopes for T cells are believed to be involved in the initiation and perpetuation of the immune response. to the protein allergen that is responsible for the clinical symptoms of the allergy. It is thought that these epitopes for T cells trigger the initial events at the level of the collaborating T cell based on attaching to an appropriate HLA molecule (HLA = human lymphocyte antigen) on the surface of a cell presenting the antigen, thereby stimulating subpopulation of T cells with the corresponding T cell receptor for the epitope. These events lead to the proliferation of T cells, the secretion of lymphokines, local inflammatory reactions, the recruitment of additional immune cells to the site of interaction between the antigen and T cells, and the activation of the B cell cascade. , leading to the production of antibodies. An isotope of these antibodies, IgE, is fundamentally important for the development of allergic symptoms, and their production is influenced early in the cascade of events at the level of the collaborating T cell by the nature of the secreted lymphokines. An epitope for T cells is the basic element, or the smallest unit of recognition by a T cell receptor, the epitope comprising essential amino acids for recognition by the receptor. Amino acid sequences that mimic those of epitopes for T cells and modify the allergic response to protein allergens are within the scope of this invention.

The selection of the peptides to distinguish those that retain the antigenic activity of Der f VII as described herein can be carried out using one or more of several different analyzes. For example, in vitro , the stimulating activity of T cells by Der f VII is analyzed based on putting a peptide known or suspected to have Der f VII antigenic activity in contact with a cell that present the antigen and present appropriate MHC molecules in a T-cell culture. The presentation of a peptide having the antigenic activity of Der f VII in association with appropriate MHC molecules to T cells in conjunction with the necessary costimulation has the effect of transmitting to the T cell a signal that induces the production of increased levels of cytokines, and particularly of interleukin-2 and interleukin-4. The culture supernatant can be obtained and analyzed to detect the presence of interleukin-2 or other known cytokines. For example, any of several conventional analyzes can be used for the determination of interleukin-2, such as the analysis described in Proc. Natl Acad. Sci. USA 86 : 1333 (1989), whose relevant parts are incorporated herein by reference. A set of materials and utensils can also be purchased from the Genzyme Corporation (Cambridge, MA) for the execution of an interferon production analysis.

Alternatively, a common analysis to determine T cell proliferation involves measuring the incorporation of tritiated thymidine. T cell proliferation can be measured in vitro by determining the amount of 3 H-labeled thymidine incorporated into the replicating DNA of the cultured cells. Accordingly, the rate of DNA synthesis can be quantified, and in turn, the rate of cell division.

In one embodiment, peptides having Der f VII T cell stimulation activity (ie the peptide comprises at least one epitope for T cells) can be identified using an algorithm that predicts the presence of epitopes for T cells in a protein sequence, such as the algorithm described by Hill et al ., Journal of Immunology , 147 : 189-197 (1991). The algorithm of Hill et al . predicts the status of epitopes for T cells in a protein by means of the presence of certain configurations within the sequence that will probably fix MHC and therefore may contain epitopes for T cells.

In order to determine the precise epitopes for T cells by, for example, precise mapping techniques, a peptide having stimulation activity of Der f VII T cells, and which therefore comprises at least one epitope for T cells according to determination by T cell biology techniques, is modified by adding or deleting amino acid residues in the amino terminal or in the carboxy terminal of the peptide, and is tested for a modification of the reactivity of the T cells to the peptide modified. Following this technique, the peptides are selected and produced by recombination or synthesis. Peptides are selected on the basis of several factors, including the strength of the response of the T cells to the peptide (eg the stimulation index), the frequency of the response of the T cells to the peptide in a population of individuals sensitive to dust mite allergens, and the potential interreactivity of the peptide with other dust mite allergens. The physical and chemical properties of these selected peptides (eg solubility and stability) are examined to determine if the peptides are suitable for use in therapeutic compositions, or if the peptides require modification as described herein. The ability of the selected peptides or of the selected modified peptides to stimulate human T cells (eg to induce lymphokine proliferation or secretion) is then determined as described herein.

In another embodiment, a peptide having the antigenic activity of Der f VII is subjected to selection to determine the ability to induce the inability of T cells to respond. The ability of a peptide known to stimulate T cells (according to has been determined by means of one or more of the analyzes described above) to inhibit or completely block the activity of the purified native Der f VII or a part thereof and to induce a state of inability to respond can be determined using subsequent attempts of stimulation of T cells with cells presenting antigen and presenting native Der f VII or peptide having the antigenic activity of Der f VII following exposure to the peptide having the antigenic activity of Der f VII. If the T cells do not respond to subsequent activation attempts, as determined by synthesis of interleukin-2 and / or proliferation of the T cells, a state of inability to respond has been induced. With respect to analysis systems that can be used as the basis for an analysis according to the present invention, see , p. eg, Gimmi et al ., (1993) Proc. Natl Acad. Sci. USA , 90 : 6586-6590, and Schwartz (1990) Science , 248 : 1349-1356.

In yet another embodiment, the peptides having the antigenic activity of Der f VII are identified by means of the IgE binding activity. For therapeutic purposes, the peptides of the invention preferably do not fix specific IgE for a dust mite allergen, or they fix such IgE to considerably lesser degree (eg at least 100 times less, and more preferably at least 1000 times less) compared to the degree to which the corresponding allergen from the purified native dust mites fixes such IgE. The reduced IgE binding activity refers to an IgE binding activity that is less than that of the purified native Der f VII protein. If a peptide having Der f VII antigenic activity must be used as a diagnostic reagent, it is not necessary for the peptide to have reduced IgE binding activity compared to the native Der f VII allergen. The IgE binding activity of the peptides can be determined by, for example, an enzyme-linked immunoassay (ELISA) using, for example, serum obtained from a subject (i.e., from an allergic subject) that has previously been exposed. native Der f VII allergen. Briefly, the peptide suspected of having Der f VII antigenic activity is applied as a coating to cavities of a microtiter culture plate. After washing and blocking the cavities, antibody solution consisting of the plasma of an allergic subject that has been exposed to a peptide suspected of having Der f VII antigenic activity is incubated in the cavities. Plasma is usually removed from IgG before incubation. A labeled secondary antibody is added to the cavities and incubated. The amount of IgE binding is then quantified and compared to the amount of IgE fixed by a purified native Der f VII protein. Alternatively, the IgE binding activity of a peptide can be determined by Western blot analysis. For example, a peptide suspected of having Der f VII antigenic activity is passed through a polyacrylamide gel using the SDS-PAGE (SDS-PAGE = characterization procedure by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate). The peptide is then transferred to nitrocellulose and is subsequently incubated with serum from an allergic subject. After incubation with a labeled secondary antibody, the amount of fixed IgE is then determined and quantified.

Another analysis that can be used to determine the IgE binding activity of a peptide is a competitive ELISA analysis. Briefly, a combination of IgE antibodies is generated based on combining plasma from subjects allergic to dust mites that have been verified by direct ELISA that have IgE that is reactive with native Der f VII. This combination is used in competitive ELISA analyzes to compare IgE binding of native Der f VII and a peptide suspected of having Der f VII activity. The binding of IgE for the native Der f VII protein and for a peptide suspected of having Der f VII antigenic activity is determined and quantified.

If a peptide that has the antigenic activity of Der f VII fixes IgE and should be used as a therapeutic agent, it is preferable that such fixation does not result in the release of mediators (eg histamines) from mast cells or basophils. To determine whether a peptide that binds IgE results in the release of mediators, a histamine release analysis can be carried out using standard reagents and protocols obtained, for example, from Amac, Inc. (Westbrook, ME). Briefly, a buffered solution of a peptide suspected of having Der f VII antigenic activity is combined with an equal volume of heparinized whole blood from an allergic subject. After mixing and incubation, the cells are granulated and supernatants are processed and analyzed using a radioimmunoassay to determine the amount of histamine released.

Peptides that have the antigenic activity of Der f VII and that should be used as therapeutic agents are preferably tested in atopy models to dust mites in mammals, such as the mouse model described in Tamura et al ., (1986) Microbiol Immunol ., 30 : 883-896, or in US Patent 4,939,239, or in the primate model described in Chiba et al ., (1990) Int. Arch. Allergy Immunol ., 93 : 83-88. Initial to determine the binding of IgE to a peptide that has the antigenic activity of Der f VII can be carried out by scratch tests or intradermal skin tests in laboratory animals or human volunteers, or in in vitro systems such as RAST (RAST = radioallergosorbent test), inhibition in RAST, ELISA analysis, RIA (radioimmunoassay), or an analysis of histamine release, as described above.

It is possible to modify the structure of a peptide that has the antigenic activity of Der f VII for purposes such as increasing solubility or enhancing therapeutic or prophylactic efficacy or stability (eg, ex vivo shelf life and resistance to proteolytic degradation in vivo ). Such modified peptides are considered functional equivalents of the peptides having the antigenic activity of Der f VII as defined herein. A modified peptide can be produced in which the amino acid sequence has been altered by procedures such as substitution, deletion or addition of amino acids, to modify immunogenicity and / or reduce allergenicity, or to which a component with The same purpose.

For example, a peptide having the antigenic activity of Der f VII may be modified to retain the ability to induce the inability of T-cells to respond and fix MHC proteins without the ability to induce a strong proliferative response or possibly any proliferative response. when administered immunogenicly. In this case, the decisive fixative residues for the receptor function of the T cells can be determined by the use of known techniques (eg the replacement of each residue and the determination of the presence or absence of reactivity of the T cells) . Those residues that have been proven to be essential for interacting with the T cell receptor can be modified by replacing the essential amino acid with another preferably similar amino acid residue (a conservative substitution) whose presence is proven to increase potency. but it does not eliminate the reactivity of the T cell or does not affect it. In addition, those amino acid residues that are not essential for interaction with the T cell receptor can be modified based on being replaced by another amino acid whose incorporation can enhance, decrease but not eliminate the reactivity of the T cell or not affect it, but do not remove the attachment to the corresponding MHC.

Additionally, a peptide having the antigenic activity of Der f VII can be modified by replacing an amino acid that has been found to be essential to interact with the MHC protein complex with another preferably similar amino acid residue (conservative substitution) whose presence it has been proven that potency decreases but does not eliminate T cell activity or does not affect it. In addition, amino acid residues that are not essential for interaction with the MHC protein complex but that however fix the MHC protein complex can be modified based on being replaced by another amino acid whose incorporation can enhance, not affect or decrease but not eliminate the reactivity of T cells. Preferred amino acid substitutions for non-essential amino acids include substitutions with alanine, glutamic acid or a methylamino acid, but are not limited thereto.

Another example of modification of a peptide having the antigenic activity of Der f VII is the substitution of cysteine residues preferably with alanine, serine, threonine, leucine or glutamic acid residues to minimize dimerization through disulfide bonds. In addition, the amino acid side chains of fragments of the protein of the invention can be chemically modified. Another modification is the cyclization of the peptide.

In order to enhance its stability and / or reactivity, a peptide having the antigenic activity of Der f VII can be modified to incorporate one or more polymorphisms in the amino acid sequence of the protein allergen resulting from any natural allelic variation. Additionally, D-amino acids, unnatural amino acids or non-amino acid analogs may be substituted or added to produce a modified protein that is within the scope of this invention. In addition, a peptide having the antigenic activity of Der f VII can be modified using polyethylene glycol (PEG) according to the method of A. Sehon et al . (Wie et al ., Supra ) to produce a protein conjugated to PEG. In addition, PEG can be added during chemical synthesis of the protein. Other modifications of a peptide having the antigenic activity of Der f VII include reduction / alkylation (Tarr, Methods of Protein Microcharacterization , JE Silver ed., Humana Press, Clifton NJ 155-194 (1986)); acylation (Tarr, supra ); chemical coupling to an appropriate support (Mishell and Shiigi, eds, Selected Methods in Cellular Immunology , WH Freeman, San Francisco, CA (1980), US Patent 4,939,239); or low concentration formalin treatment (Marsh, (1971) Int. Arch. Of Allergy and Appl. Immunol ., 41: 199 -
215).

To facilitate purification and potentially increase the solubility of a peptide having the antigenic activity of Der f VII, it is possible to add a fusion half of amino acids to the peptide backbone. For example, hexa-histidine can be added to the protein for purification by affinity chromatography of immobilized metal ions (Hochuli, E. et al ., (1988) Bio / Technology , 6 : 1321-1325). In addition, to facilitate the isolation of peptides free of irrelevant sequences, specific endoprotease segmentation sites can be introduced between the fusion half and the peptide sequences. In order to successfully desensitize a subject to the Der f VII protein allergen or the related allergen, it may be necessary to increase the solubility of the protein by adding functional groups to the protein, or by omitting hydrophobic regions of protein. Functional groups such as electrically charged amino acids and electrically charged amino acid pairs are suitable for increasing solubility when added to the amino or carboxy terminal of the peptide.

To potentially help the correct antigen processing of the epitopes for T cells within Der f VII, canonical protease-sensitive sites that each comprise at least one epitope for T cells can be introduced between regions using recombination or synthesis methods. For example, pairs of electrically charged amino acids, such as KK or RR, can be introduced between regions within a protein or a fragment during recombinant construction thereof or the same. The resulting peptide can be made sensitive to the segmentation by cathepsin and / or other trypsin-like enzymes, which would generate parts of the protein that would contain one or more epitopes for T cells. In addition, such electrically charged amino acid residues can result in an increase of the solubility of the peptide.

Site-specific mutagenesis of a nucleic acid encoding a peptide having the antigenic activity of Der f VII can be used to modify the structure of the peptide by methods known in the art. Such methods may include, among others, polymerase chain reaction (PCR) with oligonucleotide primers carrying one or more mutations (Ho et al ., (1989) Gene , 77 : 51-59) or total gene synthesis mutated (Hostomsky, Z. et al ., (1989) Biochem. Biophys. Res. Comm , 161 : 1056-1063). To enhance the expression of the recombinant protein, the aforementioned methods can be applied to change the codons present in the cDNA sequence of the invention to those preferably used by the host cell in which the recombinant protein is expressed (Wada et al . , supra ).

Another aspect of the invention is related to an antibody that is specifically reactive with a peptide having the antigenic activity of Der f VII. The antibodies of this invention can be used to standardize allergen extracts or to isolate the naturally occurring form or native form of Der f VII. For example, based on using peptides having a Der f VII activity based on the cDNA sequence of Der f VII, antiprotein / antipeptide antisera or monoclonal antibodies can be made based on using standard methods. A mammal such as a mouse, a hamster or a rabbit can be immunized with an immunogenic form of the peptide (eg Der f VII protein or an antigenic fragment that is capable of eliciting an antibody response). Techniques for conferring immunogenicity to a protein or peptide include conjugation with supports or other techniques well known in the field. A peptide having the antigenic activity of Der f VII can be administered in the presence of adjuvant. The progress of immunization can be monitored by detecting antibody titers in the plasma or in the serum. Standard ELISA immunoassay or other immunoassay can be used with the immunogen as an antigen to evaluate antibody levels.

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Following immunization, anti- Der f VII antiserum can be obtained, and, if desired, polyclonal anti- Der f VII antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be collected from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to obtain hybridoma cells. Such techniques are well known in the field, such as the hybridoma technique originally developed by Kohler and Milstein (1975) Nature , 256 : 495-497), as well as other techniques such as the human B-cell hybridoma technique (Kozbar et al ., (1983) Immunology Today , 4 : 72) and the EBV hybridoma technique (EBV = Epstein-Barr virus) to produce human monoclonal antibodies (Cole et al ., (1985) Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, Inc. pp. 77-96). The hybridoma cells can be subjected to immunochemical selection for the production of antibodies specifically reactive with a peptide having the antigenic activity of Der f VII, and the monoclonal antibodies can be isolated.

In the sense in which it is used herein, the term "antibody" is used to include fragments thereof that are also specifically reactive with the peptide having the antigenic activity of Der f VII. The antibodies can be fragmented using conventional techniques, and the fragments can be screened for their usefulness in the same manner as described above for total antibodies. For example, F (ab ') 2 fragments can be generated by treating the antibody with pepsin. The resulting F (ab ') 2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. It is further understood that the antibody of the present invention includes bispecific and chimeric molecules that have an anti- Der f VII portion.

Another aspect of this invention provides T cell clones and soluble T cell receptors specifically reactive with a peptide having the antigenic activity of Der f VII. Monoclonal T cell populations (i.e. genetically identical T cells with each other and expressing identical T cell receptors) can be obtained from an individual sensitive to Der f VII, then repeated in vitro stimulation with a protein or peptide of Der f VII having the antigenic activity of Der f VII in the presence of cells presenting the antigen in pairing with MHC. The different cells that respond to Der f VII with the MHC can then be cloned by limiting dilution, and the permanent lines can be expanded and maintained by periodic in vitro restimulation. Alternatively, Der f VII-specific hybridomas can be produced by a technique similar to that of the production of B-cell hybridomas. For example, a mammal such as a mouse is immunized with a peptide having Der f antigenic activity. VII, and the T cells are then purified and fused with a tumor cell line of autonomous growth. From the resulting hybridomas, cells that respond to a peptide having the antigenic activity of Der f VII are selected and cloned. Procedures for propagating monoclonal T cell populations are described in Cellular and Molecular Immunology (Abul K. Abbas et al . Ed.), WB Saunders Company, Philadelphia, PA (1991), page 139. Soluble T cell receptors specifically reactive with a peptide having a Der f VII activity can be obtained by immunoprecipitation using an antibody against the T cell receptor as described in Immunology: A Synthesis (Second Edition), Edward S. Golub et al ., ed., Sinauer Associates, Inc., Sunderland, MA (1991), pages 366-269.

T cell clones specifically reactive with a peptide having the antigenic activity of Der f VII can be used to molecularly isolate and clone the gene encoding the corresponding T cell receptor. In addition, a soluble T cell receptor specifically reactive with a peptide having the antigenic activity of Der f VII can be used to interfere with or inhibit antigen-dependent activation of the relevant subpopulation of T cells, for example, by administration to an individual sensitive to Der f VII. Antibodies specifically reactive with such a T-cell receptor can be produced according to the techniques described herein. Such antibodies can be used to block or interfere with the interaction of T cells with the peptides presented by
the MHC.

Exposure of allergic subjects to peptides that have the antigenic activity of Der f VII and that have the stimulatory activity of T cells may make the appropriate subpopulations of T cells unable to respond to the respective protein allergen (eg not achieving stimulate an immune response when such exposure occurs). In addition, such administration may modify the lymphokine secretion profile compared to exposure to the naturally occurring protein allergen or to a portion thereof (resulting, for example, in a decrease in IL-4 and / or a increase in IL-2). In addition, exposure to peptides that have the antigenic activity of Der f VII and that have the stimulatory activity of T cells can influence the subpopulations of T cells that normally participate in the allergen response so that these T cells are removed from the site or sites of normal exposure to the allergen (eg nasal mucosa, skin and lung) being carried to the site or sites of therapeutic administration of the protein or fragment derived therefrom. This redistribution of T-cell subpopulations can improve or reduce the ability of an individual's immune system to stimulate the usual immune response at the site of normal allergen exposure, resulting in a decrease in allergic symptoms.

When administered to a subject sensitive to dust mite allergens, a peptide having the antigenic activity of Der f VII is capable of modifying the response of B cells, the response of T cells or both the response of B cells as the response of the subject's T cells to the allergen. In the sense that the expression is used herein, the modification of the allergic response of a subject to an allergen from dust mites can be defined as an inability to respond or decrease symptoms to the allergen as determined by procedures. standard clinics (see, e.g., Varney et al ., (1990) British Medical Journal , 302 : 265-269), including a decrease in asthmatic symptoms induced by dust mites. In the sense that the expression is referred to herein, a decrease in symptoms includes any reduction of the allergic response of a subject to the allergen following a treatment regimen with a peptide of the invention. This decrease in symptoms can be determined subjectively (eg the patient feels more comfortable after being exposed to the allergen) or clinically, such as with a standard skin test.

The peptides or antibodies of the present invention can also be used to detect and diagnose Der f VII sensitivity. For example, this can be done in vitro by combining blood or blood products obtained from a subject for which the sensitivity with peptide having the antigenic activity of Der f VII should be evaluated under appropriate conditions for the fixation of blood components ( eg antibodies, T cells, B cells) with the peptide or with the peptides and on the basis of determining the degree to which such fixation takes place. Other methods of diagnosing allergic diseases in which the peptides or antibodies of the present invention can be used include the radioallergosorbent test (RAST), the radioimmunosorbent test on paper (PRIST), enzyme-linked immunoassay (ELISA), radioimmunoassays ( RIA), immunoradiometric analyzes (IRMA), luminescence immunoassays (LIA), histamine release analyzes and immunoblotting
of IgE.

The present invention also provides methods for detecting and treating the sensitivity of a subject to Der f VII. The presence in subjects of IgE specific for Der f VII and the ability of the T cells of the subjects to respond to epitopes of Der f VII for T cells can be determined by administering a Type Hypersensitivity test to the subject. Immediate and / or a Delayed Type Hypersensitivity Test ( see , e.g., Immunology (1985) Roitt, IM, Brostoff, J., Male, DK (eds), CV Mosby Co., Gower Medical Publishing, London, NY, pp. 19.2-19.18; pp. 22.1-22.10) using a peptide having the antigenic activity of Der f VII or a modified form of a peptide having the antigenic activity of Der f VII, each of which sets IgE Allergen specific. The same subjects are administered a Delayed Type Hypersensitivity test before, simultaneously with or subsequent to the administration of the Immediate Type Hypersensitivity test. Of course, if the Immediate Type Hypersensitivity test is administered before the Delayed Type Hypersensitivity test, the Delayed Type Hypersensitivity test would be administered to those subjects who had a Specific Immediate Type Hypersensitivity reaction. The Delayed Type Hypersensitivity test uses a peptide that has the antigenic activity of Der f VII, has human T cell stimulation activity and does not fix specific IgE for the allergen in a considerable percentage of the population of allergen sensitive subjects (p eg of at least about 75%). Those subjects who turn out to have both a specific Immediate Type Hypersensitivity reaction and a specific Delayed Type Hypersensitivity Reaction are administered an amount of a composition suitable for pharmaceutical administration. The composition comprises the peptide having the antigenic activity of Der f VII as used in the Delayed Type Hypersensitivity test and a pharmaceutically acceptable carrier or diluent.

A peptide having the antigenic activity of Der f VII can be used in methods to diagnose, treat or prevent allergic reactions to an allergen from dust mites or an interreactive protein allergen. Thus, the present invention provides compositions suitable for in vitro use and for pharmaceutical administration comprising an amount of at least one peptide having the antigenic activity of Der f VII. Pharmaceutical compositions will typically be formulated with a pharmaceutically acceptable carrier.

When a composition according to the invention is intended to be administered to a subject to be desensitized, such administration can be carried out using known methods and with dosages and for periods of time that are effective in reducing sensitivity (i.e., in reducing the allergic response) of the subject to an allergen from dust mites. The word "subject" is used with the intention of including living organisms in which an immune response can be provoked, such as p. ex. the mammals. Examples of subjects include humans, dogs, cats, mice, rats and transgenic species thereof. An amount of at least one peptide having the antigenic activity of Der f VII necessary to achieve a therapeutic effect may vary according to factors such as the degree of sensitivity of the subject to dust mites, age, sex and weight of the subject. , and the ability of a peptide having the antigenic activity of Der f VII to elicit an antigenic response in the subject. Dosage regimens can be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the requirements of the situation.
therapy.

The active compound (i.e., a peptide having the antigenic activity of Der f VII) can be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application or rectal administration. Depending on the route of administration, the active compound may be coated with a material to protect the compound against the action of enzymes, acids and other natural conditions that may inactivate the compound.

To administer a peptide having the antigenic activity of Der f VII by a route other than parenteral administration, it may be necessary to coat the peptide with, or co-administer the peptide with, a material to prevent its inactivation. For example, a peptide having the antigenic activity of Der f VII can be administered to an individual in an appropriate vehicle, diluent or adjuvant, or it can be co-administered with enzyme inhibitors or in an appropriate vehicle such as liposomes. Pharmaceutically acceptable diluents include saline solutions and aqueous buffer solutions. The word "adjuvant" is used in its broadest sense and includes all immune stimulating compounds such as interferon. Adjuvants contemplated herein include resorcinols and non-ionic surface agents such as polyoxyethylenelethyl ether and n-hexadecyl polyethylene ether. Enzymatic inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water CGF emulsions in oil in water, as well as conventional liposomes (Strejan et al, (1984) J. Neuroimmunol, 7:.. 27). In order to induce the inability to respond to T cells, the composition is preferably administered in a non-immunogenic manner, and e.g. ex. in a way that does not contain
adjuvant

The active compound can also be administered parenterally or intraperitoneally. The dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a condom to prevent the microorganism development

Pharmaceutical compositions suitable for injectable use include solutions (when soluble in water) or sterile aqueous dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the composition must be sterile and must be fluid to the extent necessary so that the composition can be easily injected with a syringe. The composition must be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like), suitable mixtures thereof and vegetable oils. The correct fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surface-active agents. Avoidance of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parahydroxybenzoates, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases it will be preferable to include in the composition isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol or sodium chloride. Prolonged absorption of injectable compositions can be caused by including in the composition an agent that delays absorption, such as aluminum monostearate and
jelly.

Sterile injectable solutions may be prepared based on incorporating the active agent (i.e., a peptide having the antigenic activity of Der f VII) in the amount required in an appropriate solvent with one of the ingredients or with a combination of the ingredients. listed above, as required, then sterilizing with filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients selected from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and lyophilization, which gives a powder of the active ingredient (i.e., at least one peptide having the antigenic activity of Der f VII) plus any desired additional ingredient from a solution previously filtered under conditions
sterile

When the peptide having the antigenic activity of Der f VII is adequately protected, as described above, the peptide can be administered orally, for example, with an inert diluent or an assimilable edible carrier. The peptide and the other ingredients can also be encapsulated in a hard or soft pod gelatin capsule, they can be compressed into tablets, or they can be incorporated directly into the individual's diet. For oral therapeutic administration, the active compound can be mixed with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and similar forms. The percentage of the compositions and preparations can of course be varied, and may conveniently be located between about 5 and about 80% of the weight of the unit. The amount of active component in such therapeutically useful compositions is such that an adequate dosage will be obtained.

In the sense that it is used herein, the term "pharmaceutically acceptable carrier" includes all and any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption retardants and similar materials. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional medium or agent is incompatible with the active compound, the use thereof in the therapeutic compositions is contemplated. They can also be incorporated into the active compound compositions
supplementary

It is especially advantageous to formulate parenteral compositions in the form of dosage units for easy administration and to achieve a uniformity of dosage. In the sense in which it is used herein, the expression "in the form of dosage units" refers to to physically discrete units suitable as dosages units for mammalian subjects to be treated, containing each unit a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the Pharmaceutical vehicle required. The specifications for dosage unit forms of the invention come determined by and are directly dependent on (a) the unique characteristics of the active compound and the concrete effect therapeutic to achieve, and (b) the limitations inherent in the technique of combining such active compound for the treatment of sensitivity in the subjects

The present invention also provides a composition comprising at least two peptides that have the antigenic activity of Der f VII (eg a physical mixture of at least two peptides) and that each have T cell stimulation activity. For example, at least two peptides each having an antigenic activity of Der f VII can be combined. Alternatively, a peptide having at least two regions each having a T cell stimulation activity (i.e., a peptide in which each such region comprises at least one epitope for cells) can be administered to an allergic subject T). Such a peptide may have at least two regions derived from the same Der f VII allergen. A composition of two peptides or a peptide having at least two regions may be administered to a subject, in the form of a composition with a pharmaceutically acceptable carrier as described above. It may be administered simultaneously or sequentially to an allergen sensitive subject of dust mites to treat such sensitivity an amount of one or more such compositions. Such compositions may be useful for the manufacture of a medicament for treating the sensitivity to house dust mites in an individual.

The cDNA (or mRNA that served as a template during reverse transcription) that encodes a peptide that has the antigenic activity of Der f VII can be used to identify similar nucleic acid sequences in any variety or type of animal, and therefore to Molecularly cloning genes that have sufficient sequential homology to hybridize with the cDNA encoding a peptide that has the antigenic activity of Der f VII. Accordingly, the present invention includes not only peptides having the antigenic activity of Der f VII, but also other proteins that can be DNA-encoded allergens that hybridize with DNA of the present invention.

Within the scope of the invention are isolated peptides that are immunologically related to Der f VII such as by interreactivity with antibodies or by interreactivity with T cells, and which are distinct from those already identified. Such peptides bind the antibodies specific for the protein and peptides of the invention, or stimulate the T cells specific for the protein and peptides of this invention.

A peptide having the antigenic activity of Der f VII (i.e. Der f VII produced by recombination or chemical synthesis) is exempt from all other dust mite proteins, and is therefore useful in the standardization of extracts of allergens that are key reagents for the diagnosis and treatment of hypersensitivity to dust mites. In addition, such a peptide is of a coherent and well defined composition and biological activity to be used in preparations that can be administered for therapeutic purposes (eg to modify the allergic response and a subject sensitive to dust mites). Such peptides can also be used to study the immunotherapy mechanism of Dermatophagoides farinae allergy and to design derivatives or modified analogs useful in immunotherapy.

The work carried out by third parties has shown that generally the best results during immunotherapy (that is, the best relief of symptoms) are obtained with high doses of allergen extracts. However, many subjects are unable to tolerate large doses of such extracts due to systemic reactions caused by allergens and the other components that are in these preparations. A peptide having the antigenic activity of Der f VII according to the invention has the advantage of being free of all other dust mite proteins, and therefore is more harmless and more suitable for applications.
therapeutic

It is now also possible to design an agent or drug capable of blocking or inhibiting the ability of an allergen from dust mites to induce an allergic reaction in sensitive subjects. Such agents could be designed, for example, in such a way that they were fixed to the corresponding anti- Der f VII molecules, thus preventing the fixation of the allergen and the IgE and the subsequent degranulation of the mast cells / basophils. Alternatively, such agents could be fixed to cellular components of the immune system, resulting in a suppression or desensitization of allergic responses to dust mite allergens. A non-limiting example of this is the use of peptides that include epitopes of Der f VII for B or T cells, or modifications thereof, based on the protein structure of the Der f VII cDNA to suppress the allergic response to an allergen from dust mites. This could be done by defining the structures of the fragments encoding the epitopes for B and T cells that affect the functioning of B and T cells in in vitro studies with blood components of dust-sensitive mite-sensitive subjects.

The invention is further illustrated by the following examples, which should not be construed as examples that further limit the present invention. The contents of all references and published patent applications cited within this entire application are incorporated herein by reference.
rencia.

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Example 1 Isolation of the cDNA clone encoding Der f VII from a cDNA library of? Lambt11

A λdagt11 cDNA library was prepared from Dermatophagoides farinae live adults acquired from the Fa. Commonwealth Serum Laboratories, Parkville, Australia (Thomas, W., et al ., Int Arch Allergy Appl Immunol (1988) 85 : 127-9). The library was prepared according to Trudinger et al ., (1991) Chem. Exp. Allergy , 21: 33-37.

PCR amplification and DNA sequencing were used to isolate Der f VII cDNA from the λ1 library. An oligonucleotide primer (Df1 in Table 1) was performed based on the expected N-terminal sequence of Der p VII. Said primer had the sequence GCGAATTCGATCCAATTCACTATGAT-3 '(SEQ ID NO: 8). The first GCGAATTC encodes an EcoRI site (GAATTC) and the GAT sequence encodes the first six residues of Der p VII. For the other primer the direct primer of λ GGTGGCGACGACTCCTGGAGCCCG-3 '(SEQ ID NO: 9) (Df2 in Table 1) was used next to the EcoRI cloning site (New England Biolabs, Beverly, United States).
United).

The PCR reactions were carried out in a final 50 µl reaction volume containing 20 mM Tris-HCl at pH 8.2, 10 mM KCl, 6 mM (NH 4) 2 SO 4 2 mM MgCl 2, 0.1% Triton X-100, 10 ng of nuclease free BSA, 10 mM dNTPs, 20 pmol of each primer and 2.5 units of Pfu DNA polymerase. This was obtained as a set of Stratagene (La Jolla, California, United States). Target DNA (cDNA bonds of λD farinae , 0.001 µg) was added and the contents of the tube were mixed and covered with paraffin oil. The tubes were initially denatured at 95 ° C for 5 minutes, then tempered at 55 ° C for 2 minutes and expanded at 72 ° C for 2 minutes. After this, denaturation was performed at 94 ° C for 1 minute, cooling at 55 ° C for 1 minute and expansion as before, over 48 cycles. In the final cycle (number 50), the expansion reaction increased to 10 minutes to ensure that all amplified products were at their maximum
length.

Next, amplified bands were searched per 10 microliters of the reaction, in a 1% agarose gel. He leftover from the reaction mixture was ethanol precipitated before the Purification of the amplified product on a low agarose gel melting point (Bio-Rad., Richmond, United United).

The purified PCR product was digested with EcoRI and ligated to the M18 mp18 vector (see Messing, supra ), digested with EcoRI and then transferred to competent E. coli cells of strain TG1. Isolated white plates were taken and used to prepare phage stores and single stranded DNA for sequencing.

Example 2 DNA sequence analysis of Der f VII cDNA

DNA sequencing was carried out using the dideoxynucleotide chain termination method, using version 2.0 of Sequenase (USB Corp., Cleveland, United States) according to the supplier's protocol. Among the primers used for sequencing, mention should be made of the M13 sequencing primer (-40) at 17-mer GTTTTCCCAGTCACGAC-3 '(SEQ ID No. 10) (Df3 in Table 1), the Df1 primer (SEQ ID No. 8) used for the PCR reaction described in example 1 and two other oligonucleotide primers, Df4 and Df5, both shown in Table 1. Df4 primer GGTGAATTAGCCATGCG-3 '(SEQ ID No. 11) was previously used for sequencing Der p VII and primer Df5 TCAATCTTGGATCCAATTTTTGGC-3 '(SEQ ID No. 12) was based on Der f VII sequences of nucleotides 559-582.

To isolate a cDNA containing the untranslated region of the 5 'side of Der f VII, an oligonucleotide primer based on the C-terminal sequence of Der f VII was produced. Said primer (Df6 in Table 1) had the sequence GGAATTCTTAATTTTTTTCCAATTCACG-3 '(SEQ ID NO: 13). The first GGAATTC encodes an EcoRI site. Said sequence and the following one (TTA ...) are complementary to the reverse sequences of the stop codon and the last six residues of Der f VII. For the other primer the reverse primer of? Lambdagt11 TTGACACCAGACCAACTGG was used
TAATG-3 '(SEQ ID No. 14) (Df7 in Table 1), next to the EcoRI cloning site.

The PCR reactions were carried out according to the conditions described in example 1. The PCR product was purified on a low melting agarose gel, digested with EcoRI and ligated to pUC 19 digested with EcoRI, and then transferred to competent E. coli cells of strain TG1. Transformative E. coli plasmid DNA was isolated and used for sequencing.

DNA sequencing was carried out by the same dideoxynucleotide chain termination method, using version 2.0 of Sequenase, described above. However, before sequencing, the double-chain plasmid DNA templates were denatured, by treatment with NaOH, and neutralized by the addition of sodium acetate and ethanol precipitated according to the supplier's protocol. To obtain the untranslated region of the 5 'side of Der f VII cDNA, primer Df8 was used (see Table 1). Said primer had the sequence ATGACGTTCGAATTTATC-3 '(SEQ ID No. 15), which corresponds to the inverse sequence of Der f VII of nucleotide No. 225-208.

TABLE 1 Oligonucleotides used for amplification and PCR sequencing

Name Sequence Derived from positions nucleotide of Der f VII Df1 GCGAATTCGATCCAATTCACTATGAT-3 ' 94-111 Df2 GGTGGCGACGACTCCTGGAGCCCG-3 ' primer direct \ lambdagt11 Df3 GTTTTCCCAGTCACGAC-3 ' primer sequencing M13 (-40) Df4 GGTGAATTAGACATGCG-3 ' 247-263 Df5 TCAATCTTGGATCCAATTTTTGGC-3 ' 559-582 Df6 CGAATTCTTAATTTTTTTCCAATTCACG-3 ' 684-664 Df7 TTGACACCAGACCAACTGGTAATG-3 ' primer direct \ lambdagt11 Df8 ATGACGTTCGAATTTATC-3 ' 225-208

Equivalent

Those skilled in the art may recognize or distinguish, using only routine experimentation, numerous equivalents to the specific embodiments that have been described in this document. It is considered that said equivalents are within the scope of the present invention and covered by the appended claims.

(1. General Information:

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(i)

Applicant

           \ vskip0.500000 \ baselineskip
        

(TO)

Name: Western Australian Research Institute for Child Health

           \ vskip0.500000 \ baselineskip
        

(B)

GPO Box D184 Street

           \ vskip0.500000 \ baselineskip
        

(C)

City: Perth

           \ vskip0.500000 \ baselineskip
        

(D)

State: MA

           \ vskip0.500000 \ baselineskip
        

(AND)

    Country: Western Australia

           \ vskip0.500000 \ baselineskip
        

(F)

    Postal Code (ZIP): 6001

           \ vskip0.800000 \ baselineskip
        

(ii)

  Title of the invention:

           \ vskip0.800000 \ baselineskip
        

Protein and allergenic peptides of house dust mites and applications for them

           \ vskip0.800000 \ baselineskip
        

(iii)

  Number of sequences: 15

           \ vskip0.800000 \ baselineskip
        

(iv)

Postal Address:

           \ vskip0.500000 \ baselineskip
        

(TO)

   Recipient: Lahive & Cockfield

           \ vskip0.500000 \ baselineskip
        

(B)

   Address: 60 State Street

           \ vskip0.500000 \ baselineskip
        

(C)

   Location: Boston

           \ vskip0.500000 \ baselineskip
        

(D)

   State: MA

           \ vskip0.500000 \ baselineskip
        

(AND)

    Country: United States

           \ vskip0.500000 \ baselineskip
        

(F)

    Postal Code (ZIP): 02109

           \ vskip0.800000 \ baselineskip
        

(v)

  Computer reading form:

           \ vskip0.500000 \ baselineskip
        

(TO)

Media Type: Floppy Disk

           \ vskip0.500000 \ baselineskip
        

(B)

Computer: IBM PC compatible

           \ vskip0.500000 \ baselineskip
        

(C)

Operating system: PC-DOS / MS-DOS

           \ vskip0.500000 \ baselineskip
        

(D)

Program: PatentIn edition n ° 1.0, version n ° 1.25

           \ vskip0.800000 \ baselineskip
        

(saw)

Data on the current application:

           \ vskip0.500000 \ baselineskip
        

(TO)

   Application number:

           \ vskip0.500000 \ baselineskip
        

(B)

   Application date:

           \ vskip0.500000 \ baselineskip
        

(C)

   Classification:

           \ vskip0.800000 \ baselineskip
        

(vii)

Data on previous requests:

           \ vskip0.500000 \ baselineskip
        

(TO)

Application number: USSN 08/031 141

           \ vskip0.500000 \ baselineskip
        

(B)

Application date: March 12, 1993

           \ vskip0.500000 \ baselineskip
        

(C)

Application number: USSN 08/081 540

           \ vskip0.500000 \ baselineskip
        

(D)

Application date: June 22, 1993

           \ vskip0.800000 \ baselineskip
        

(viii)

Information about him agent:

           \ vskip0.500000 \ baselineskip
        

(TO)

   Name: Amy E. Mandragouras

           \ vskip0.500000 \ baselineskip
        

(B)

   Registration number: 36,207

           \ vskip0.500000 \ baselineskip
        

(C)

   Reference number: 053.2 PCT (IMI-032CPPC)

           \ vskip0.800000 \ baselineskip
        

(ix)

Contact information:

           \ vskip0.500000 \ baselineskip
        

(TO)

   Phone: 617-227-7400

           \ vskip0.500000 \ baselineskip
        

(B)

   Fax: 617-227-5941

           \ vskip1.000000 \ baselineskip
        

(2) Information for SEQ ID NO: 1:

           \ vskip0.800000 \ baselineskip
        

(i)

   Sequence characteristics:

           \ vskip0.500000 \ baselineskip
        

(TO)

Length: 812 base pairs

           \ vskip0.500000 \ baselineskip
        

(B)

Type: nucleic acid

           \ vskip0.500000 \ baselineskip
        

(C)

Number of strands: one strand

           \ vskip0.500000 \ baselineskip
        

(D)

Topology: linear

           \ vskip0.800000 \ baselineskip
        

(ii)

Molecule Type: cDNA

           \ vskip0.800000 \ baselineskip
        

(ix)

Features:

           \ vskip0.500000 \ baselineskip
        

(TO)

Name / key: CDS

           \ vskip0.500000 \ baselineskip
        

(B)

Location: 68..712

           \ vskip0.800000 \ baselineskip
        

(xi)

Description of the sequence SEQ ID NO: 1:

one

2

           \ vskip1.000000 \ baselineskip
        

(2) Information for SEQ ID NO: 2:

           \ vskip0.800000 \ baselineskip
        

(i)

     Sequence characteristics:

           \ vskip0.500000 \ baselineskip
        

(TO)

   Length: 215 amino acids

           \ vskip0.500000 \ baselineskip
        

(B)

    Type: amino acid

           \ vskip0.500000 \ baselineskip
        

(C)

        Topology: linear

           \ vskip0.800000 \ baselineskip
        

(ii)

    Molecule Type: Protein

           \ vskip0.800000 \ baselineskip
        

(xi)

   Sequence description: SEQ ID NO: 2:

3

4

           \ vskip1.000000 \ baselineskip
        

(2) Information for SEQ ID NO: 3:

           \ vskip0.800000 \ baselineskip
        

(i)

   Sequence characteristics:

           \ vskip0.500000 \ baselineskip
        

(TO)

     Length: 18 base pairs

           \ vskip0.500000 \ baselineskip
        

(B)

      Type: nucleic acid

           \ vskip0.500000 \ baselineskip
        

(C)

      Number of strands: of a strand

           \ vskip0.500000 \ baselineskip
        

(D)

     Topology: linear

           \ vskip0.800000 \ baselineskip
        

(ii)

    Molecule Type: cDNA

           \ vskip0.800000 \ baselineskip
        

(xi)

   Description of the sequence SEQ ID NO: 3:

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

\hskip-.1em\dddseqskip

GATCCAATTC ACTATGAT

\hfill

18

 \ hskip-.1em \ dddseqskip 

GATCCAATTC ACTATGAT

 \ hfill 

18

           \ vskip1.000000 \ baselineskip
        

(2) Information for SEQ ID NO: 4:

           \ vskip0.800000 \ baselineskip
        

(i)

Sequence characteristics:

           \ vskip0.500000 \ baselineskip
        

(TO)

Length: 17 base pairs

           \ vskip0.500000 \ baselineskip
        

(B)

Type: nucleic acid

           \ vskip0.500000 \ baselineskip
        

(C)

Number of strands: of a strand.

           \ vskip0.500000 \ baselineskip
        

(D)

Topology: linear

           \ vskip0.800000 \ baselineskip
        

(ii)

    Molecule Type: cDNA

           \ vskip0.800000 \ baselineskip
        

(xi)

   Sequence description: SEQ ID NO: 4:

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

\hskip-.1em\dddseqskip

GGTGAATTAG ACATGCG

\hfill

17

 \ hskip-.1em \ dddseqskip 

GGTGAATTAG ACATGCG

 \ hfill 

17

           \ vskip1.000000 \ baselineskip
        

(2) Information for SEQ ID NO: 5:

           \ vskip0.800000 \ baselineskip
        

(i)

Characteristics of the sequence:

           \ vskip0.500000 \ baselineskip
        

(TO)

Length: 24 base pairs

           \ vskip0.500000 \ baselineskip
        

(B)

Type: nucleic acid

           \ vskip0.500000 \ baselineskip
        

(C)

Number of strands: of a strand

           \ vskip0.500000 \ baselineskip
        

(D)

Topology: linear

           \ vskip0.800000 \ baselineskip
        

(ii)

  Molecule Type: cDNA

           \ vskip0.800000 \ baselineskip
        

(xi)

  Sequence description: SEQ ID NO: 5:

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

\hskip-.1em\dddseqskip

TCAATTTTGG ATCCAATTTT CGCT

\hfill

24

 \ hskip-.1em \ dddseqskip 

TCAATTTTGG ATCCAATTTT CGCT

 \ hfill 

24

           \ vskip1.000000 \ baselineskip
        

(2) Information for SEQ ID NO: 6:

           \ vskip0.800000 \ baselineskip
        

(i)

Sequence characteristics:

           \ vskip0.500000 \ baselineskip
        

(TO)

Length: 761 base pairs

           \ vskip0.500000 \ baselineskip
        

(B)

Type: nucleic acid

           \ vskip0.500000 \ baselineskip
        

(C)

Number of strands: one strand

           \ vskip0.500000 \ baselineskip
        

(D)

Topology: linear

           \ vskip0.800000 \ baselineskip
        

(ii)

Molecule Type: cDNA

           \ vskip0.800000 \ baselineskip
        

(ix)

Features:

           \ vskip0.500000 \ baselineskip
        

(TO)

Name / key: CDS

           \ vskip0.500000 \ baselineskip
        

(D)

Location: 43… 681

           \ vskip0.800000 \ baselineskip
        

(xi)

Sequence description ID SEC Nº: 6:

           \ vskip1.000000 \ baselineskip
        

5

6

           \ vskip1.000000 \ baselineskip
        

(2) Information for SEQ ID NO: 7:

           \ vskip0.800000 \ baselineskip
        

(i)

Characteristics of the sequence:

           \ vskip0.500000 \ baselineskip
        

(TO)

Length: 213 amino acids

           \ vskip0.500000 \ baselineskip
        

(B)

Type: amino acid

           \ vskip0.500000 \ baselineskip
        

(AND)

Topology: linear

           \ vskip0.800000 \ baselineskip
        

(ii)

  Molecule Type: Protein

           \ vskip0.800000 \ baselineskip
        

(xi)

Sequence description: SEQ ID NO: 7:

7

           \ vskip1.000000 \ baselineskip
        

(2) Information for SEQ ID NO: 8:

           \ vskip0.800000 \ baselineskip
        

(i)

Sequence characteristics:

           \ vskip0.500000 \ baselineskip
        

(TO)

Length: 26 base pairs

           \ vskip0.500000 \ baselineskip
        

(B)

Type: nucleic acid

           \ vskip0.500000 \ baselineskip
        

(C)

Number of strands: of a strand

           \ vskip0.500000 \ baselineskip
        

(D)

Topology: linear

           \ vskip0.800000 \ baselineskip
        

(ii)

Molecule Type: cDNA

           \ vskip0.800000 \ baselineskip
        

(xi)

Description of the sequence SEQ ID NO: 8:

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

\hskip-.1em\dddseqskip

GCGAATTCGATCCAATTCACTATGAT

\hfill

26

 \ hskip-.1em \ dddseqskip 

GCGAATTCGATCCAATTCACTATGAT

 \ hfill 

26

           \ vskip1.000000 \ baselineskip
        

(2) Information for SEQ ID NO: 9:

           \ vskip0.800000 \ baselineskip
        

(i)

Characteristics of the sequence:

           \ vskip0.500000 \ baselineskip
        

(TO)

Length: 24 base pairs

           \ vskip0.500000 \ baselineskip
        

(B)

  Type: nucleic acid

           \ vskip0.500000 \ baselineskip
        

(C)

      Number of strands: of a strand

           \ vskip0.500000 \ baselineskip
        

(D)

     Topology: linear

           \ vskip0.800000 \ baselineskip
        

(ii)

    Molecule Type: cDNA

           \ vskip0.800000 \ baselineskip
        

(xi)

   Description of the sequence SEQ ID NO: 9:

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

\hskip-.1em\dddseqskip

GGTGGCGACGACTCCTGGAGCCCG

\hfill

24

 \ hskip-.1em \ dddseqskip 

GGTGGCGACGACTCCTGGAGCCCG

 \ hfill 

24

           \ vskip1.000000 \ baselineskip
        

(2) Information for SEQ ID NO: 10:

           \ vskip0.800000 \ baselineskip
        

(i)

   Sequence characteristics:

           \ vskip0.500000 \ baselineskip
        

(TO)

     Length: 17 base pairs

           \ vskip0.500000 \ baselineskip
        

(B)

      Type: nucleic acid

           \ vskip0.500000 \ baselineskip
        

(C)

      Number of strands: of a strand

           \ vskip0.500000 \ baselineskip
        

(D)

     Topology: linear

           \ vskip0.800000 \ baselineskip
        

(ii)

    Molecule Type: cDNA

           \ vskip0.800000 \ baselineskip
        

(xi)

   Description of the sequence SEQ ID NO: 10:

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

\hskip-.1em\dddseqskip

GTTTTCCCAGTCACGAC

\hfill

17

 \ hskip-.1em \ dddseqskip 

GTTTTCCCAGTCACGAC

 \ hfill 

17

           \ vskip1.000000 \ baselineskip
        

(2) Information for SEQ ID NO: eleven:

           \ vskip0.800000 \ baselineskip
        

(i)

   Sequence characteristics:

           \ vskip0.500000 \ baselineskip
        

(TO)

     Length: 17 base pairs

           \ vskip0.500000 \ baselineskip
        

(B)

      Type: nucleic acid

           \ vskip0.500000 \ baselineskip
        

(C)

      Number of strands: of a strand

           \ vskip0.500000 \ baselineskip
        

(D)

     Topology: linear

           \ vskip0.800000 \ baselineskip
        

(ii)

    Molecule Type: cDNA

           \ vskip0.800000 \ baselineskip
        

(xi)

   Description of the sequence SEQ ID NO: 11:

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

\hskip-.1em\dddseqskip

GGTGAATTAGACATGCG

\hfill

17

 \ hskip-.1em \ dddseqskip 

GGTGAATTAGACATGCG

 \ hfill 

17

           \ vskip1.000000 \ baselineskip
        

(2) Information for SEQ ID NO: 12:

           \ vskip0.800000 \ baselineskip
        

(i)

Characteristics of the sequence:

           \ vskip0.500000 \ baselineskip
        

(TO)

     Length: 24 base pairs

           \ vskip0.500000 \ baselineskip
        

(B)

      Type: nucleic acid

           \ vskip0.500000 \ baselineskip
        

(C)

      Number of strands: of a strand

           \ vskip0.500000 \ baselineskip
        

(D)

     Topology: linear

           \ vskip0.800000 \ baselineskip
        

(ii)

    Molecule Type: cDNA

           \ vskip0.800000 \ baselineskip
        

(xi)

   Description of the sequence SEQ ID NO: 12:

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

\hskip-.1em\dddseqskip

TCAATCTTGGATCCAATTTTTGGC

\hfill

24

 \ hskip-.1em \ dddseqskip 

TCAATCTTGGATCCAATTTTTGGC

 \ hfill 

24

           \ vskip1.000000 \ baselineskip
        

(2) Information for SEQ ID NO: 13:

           \ vskip0.800000 \ baselineskip
        

(i)

   Sequence characteristics:

           \ vskip0.500000 \ baselineskip
        

(TO)

     Length: 24 base pairs

           \ vskip0.500000 \ baselineskip
        

(B)

      Type: nucleic acid

           \ vskip0.500000 \ baselineskip
        

(C)

      Number of strands: of a strand

           \ vskip0.500000 \ baselineskip
        

(D)

     Topology: linear

           \ vskip0.800000 \ baselineskip
        

(ii)

    Molecule Type: cDNA

           \ vskip0.800000 \ baselineskip
        

(xi)

   Description of the sequence SEQ ID NO: 13:

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

\hskip-.1em\dddseqskip

GGAATTCTTAATTTTTTTCCAATTCACG

\hfill

28

 \ hskip-.1em \ dddseqskip 

GGAATTCTTAATTTTTTTCCAATTCACG

 \ hfill 

28

           \ vskip1.000000 \ baselineskip
        

(2) Information for SEQ ID NO: 14:

           \ vskip0.800000 \ baselineskip
        

(i)

Characteristics of the sequence:

           \ vskip0.500000 \ baselineskip
        

(TO)

     Length: 24 base pairs

           \ vskip0.500000 \ baselineskip
        

(B)

      Type: nucleic acid

           \ vskip0.500000 \ baselineskip
        

(C)

      Number of strands: of a strand

           \ vskip0.500000 \ baselineskip
        

(D)

     Topology: linear

           \ vskip0.800000 \ baselineskip
        

(ii)

    Molecule Type: cDNA

           \ vskip0.800000 \ baselineskip
        

(xi)

   Description of the sequence SEQ ID NO: 14:

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

\hskip-.1em\dddseqskip

TTGACACCAGACCAACTGGTAATG

\hfill

24

 \ hskip-.1em \ dddseqskip 

TTGACACCAGACCAACTGGTAATG

 \ hfill 

24

           \ vskip1.000000 \ baselineskip
        

(2) Information for SEQ ID NO: 15:

           \ vskip0.800000 \ baselineskip
        

(i)

   Sequence characteristics:

           \ vskip0.500000 \ baselineskip
        

(TO)

     Length: 24 base pairs

           \ vskip0.500000 \ baselineskip
        

(B)

      Type: nucleic acid

           \ vskip0.500000 \ baselineskip
        

(C)

      Number of strands: of a strand

           \ vskip0.500000 \ baselineskip
        

(D)

     Topology: linear

           \ vskip0.800000 \ baselineskip
        

(ii)

    Molecule Type: cDNA

           \ vskip0.800000 \ baselineskip
        

(xi)

   Description of the sequence SEQ ID NO: 15:

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

\hskip-.1em\dddseqskip

ATGACGTTCGAATTTATC

\hfill

18

 \ hskip-.1em \ dddseqskip 

ATGACGTTCGAATTTATC

 \ hfill 

18

Claims (17)

1. Isolated nucleic acid comprising a nucleotide sequence encoding a Der f VII protein allergen comprising the amino acid sequence of SEQ ID NO: 7 or the mature portion of said allergen. 2. Isolated nucleic acid comprising a nucleotide sequence encoding a Der f VII protein allergen comprising a portion of the amino acid sequence of SEQ ID NO: 7, wherein said portion does not undergo a cross reaction with the protein allergen Der p VII. 3. Isolated nucleic acid according to claim 1 or 2, comprising the nucleotide sequence of SEQ ID NO: 6. 4. Isolated nucleic acid according to claim 3, comprising the coding region of the nucleotide sequence of SEQ ID NO: 6. 5. Isolated nucleic acid according to claim 1 or 2, wherein the protein allergen comprises the amino acid sequence of SEQ ID NO: 7. 6. Isolated nucleic acid according to claim 1 or 2, wherein the peptide has a length of at minus 10-20 amino acids. 7. Isolated nucleic acid according to claim 1 or 2, wherein the peptide has a length of at minus 10-16 amino acids. 8. Recombinant expression vector that comprises the nucleic acid of any one of claims 1 to 7. 9. Host cell transfected with the vector of recombinant expression of claim 8. 10. Method for producing a Der f VII protein allergen or a peptide comprising at least one epitope for T cells thereof, said method comprising the steps of culturing the host cell of claim 8 in medium to express the protein allergen or peptide, and isolate the protein allergen or peptide from the culture. 11. Protein allergen of Der f VII of Group VIII isolated comprising the amino acid sequence of SEQ ID NO: 7, or the mature portion thereof. 12. Protein allergen of Der f VII of Group VIII isolated comprising the amino acid sequence of SEQ ID NO: 7, wherein said portion does not undergo a cross reaction with the protein allergen Der p VII. 13. Allergen or protein peptide according to claim 11 or 12, produced by recombinant expression of a nucleic acid encoding the protein allergen or peptide. 14. Allergen or protein peptide according to claim 11 or 12, produced by chemical synthesis. 15. Allergen or protein peptide according to claim 11 or 12, which is a length of at least 10-20 amino acids 16. Preparation in pure substance of an allergen or protein peptide according to any of claims 11 to fifteen. 17. Allergen or protein peptide according any of claims 11 to 15 for the treatment or the diagnosis of the sensitivity of an individual to the mites of the powder.